Physiology 735 –lab 4

Recording Gross potentials.

Friday: Oct. 9, 2004

Check:  http://www.physiology.wisc.edu/phys735/labs/ for the latest.

 

The goal is to become familiar with some of the techniques of recording gross potentials in the auditory system. This involves the use of (relatively) large electrodes, usually made out of silver wire because of its low resistance and resistance to corrosion and more importantly, it can be chlorided (silver converted to silver chloride = AgCl) so that the electrode can either accept or lend an electron while passing current.

Surgery: A chinchilla will be prepared for recording: this involves anesthetization with sodium pentobarbital, dose rate of  ~75mg/kg of body weight. Additional doses are given at roughly 10% of the anesthetic dose and determined by whether the animal responds to a paw pinch by withdrawing its leg (your responsibility). Its respiratory pattern is also watched (if the rate increases then give an additional dose. If it is irregular/ intermittent, no additional dose as the animal could be expiring. A tracheotomy is performed to reduce the likelihood of the respiratory airway becoming obstructed during the experiment. The animal's temperature is kept at 37°C through monitoring with a rectal probe (thermometer) and a homeothermic (heating) blanket on which the animal rests.

The animal's skull is cleaned in order to accept a head mount to which a post is attached. The bulla is then exposed and an opening is made so that the round window can be visualized. A wire is then positioned so that it contacts the round window. The wire is cemented to the bulla. A hypodermic needle (or an alligator clip) serves as a reference electrode and is positioned in a jaw muscle. These wires go to a preamplifier which acts as a buffer (to other electronics) and also reduces the noise that would otherwise be present at the input to the computer A/D system. The opening into the bulla is then sealed after a vent tube is placed in it.

Have someone speak into the open meatus and observe the signal on an oscilloscope.

An earphone is coupled to the meatus and a probe tube microphone is inserted (Etymotic ER10C).  A gain of 20 dB is selected on the microphone amplifier.

Exit windows….Click on Start.  Select restart in DOS mode.  When the prompt comes up…    type c:\syst (enter).    Then syst4 (enter).    To activate a ‘macro command’  type m then the macro name. you hacircuit diagram for the control unit on the ST110HBD Sta-rite system for spase to repeat this after you compute the calibration curve for the phone.

v    The phone is calibrated using a program called earcal4.

v    This program presents short tones at increasing frequencies,

v    It samples the output of a microphone sensing the pressure at the eardrum of the subject,

v    Then averages the response,

v    after all the frequencies have been presented… exit the data collection program (SYST).

v    ESC then exit to DOS.

v    Type WIN.

v    Startup MATLAB.

v    Type scomptab(RTN) (to compute a correction curve that will be used for the data collection portion of the experiment.

v    You have to provide the animal ID: lab4.

The microphone and probe tube have been previously calibrated so that the sound pressure level in dB per Volts at the input to the A/D is known.

v    The calibration is to be made from 100 to 18,000 Hz in 100 Hz steps.

v    The resulting amplitude plot corresponds to the sound pressure level (dB re 20 mPa) for 0 dB attenuation.

v    The attenuation covers a 0 to 120 dB range in 1 dB steps.

·       plot the calibration.

·      This calibration will also be stored in the computer for use in the data collection process (so the amplitude can be corrected for the phone characteristic): pick an SPL that covers the range out to 14 or 16 kHz. One asked, give the result a name like f95 if the level chosen was 95 dB SPL. You need to pick the highest level that cover all (most) of the frequency range of interest). This name will be used by the data collection program (SYST4). After returning to the data collection program… type C to enter in the compensation curve. Then m. then caplab4.

Data collection.

Cochlear microphonic potentials.

One of the first potentials ever recorded in the auditory system was the cochlear microphonic (CM). This is the integrated output of some portion of the cochlea generated primarily by the OHCs (outer hair cells). CM has no threshold. It will appear in the signal of other recordings just because it can be rather large - millivolts. At times you may have to take measures to eliminate it from recordings when you are not interested in it. CM can also be used as a measure of the sound transmission through the middle ear.

 

One can use the caplab4 program to record CM and EPs over a “response space – a two-dimensional space with frequency and intensity as the parameters” that you specify:

·       Try 1000 to 20000Hz in 1000Hz increments: 0 to 80 dB SPL in 10 dB increments. Set the gain so that there is no clipping of the signal at the highest level.

Since the CM follows the motion of the basilar membrane (remember Dallos' hair cell studies), the waveform will invert if you invert the polarity of the acoustic signal. This fact has been used when you want to focus on N1 potentials. That is, if you alternate the polarity of the acoustic signal the CM will cancel upon averaging while the N1 potential will become more prominent. Try it. Use 2000 Hz and 70 dB SPL and type w to set the number of reps and alternate polarity.

N1 Potentials.

One commonly used method of monitoring the viability of the cochlea during an experiment is to determine the N1 threshold using visual detection criteria. The N1 is the integrative signal produced by the auditory nerve due to a synchronous firing in response to an acoustic signal. A click works best but has a limitation since it excites the entire cochlea and hence all auditory nerve fibers. The modification that is used in order to ascertain the function of separate regions of the cochlea is to shape (turn the tone on/off with a specified rise/fall time) a short tone pulse (at different frequencies) and present it enough times to obtain a reliable average. At sufficiently low levels only a relatively restricted region of the cochlea is excited, hence, one can obtain the N1 threshold in a frequency specific manner. The threshold data becomes a reference for future use: if after further experimental manipulation or just the passage of time resulting in a “rundown” of the preparation, the thresholds have risen, then one makes a note of it and if the change is larger than can be accepted, the experiment may have to be terminated.

·       Repeat the data collection for 2000 Hz to 14000Hz, 10 to 60 dB SPL with 5 dB increments and with the alternate polarity option selected. Type W for sweeps  20 alternate.

·       Obtain the N1 response at 500 Hz and 1000 Hz. Vary the intensity from 30 to 80 dB SPL. Note that it is more difficult to obtain N1 at low frequencies. Why?

·       How is the response different from the response to higher frequencies?

·       How does the N 1 latency vary as a function of frequency and SPL?

ABR  Definitions:

ABR (auditory brainstem recording) is a far field potential: the recording electrodes are distant from the generators in the brainstem. The peaks in the resulting waveform are numbered using the Roman numerals. In particular, wave V corresponds to a source in or near the IC.

AEPs (auditory evoked potentials) were recorded in the 1930's. The recordings were largest when the electrodes were on the vertex. There are late potentials that occur with a latency of 50 to 200ms.

AEPs result from both synchronized neural discharge patterns and graded post-synaptic potentials and are of clinical significance, e.g., detection of acoustic tumors.

CM: primary receptor potential. There is no threshold for CM recordings.

SP: 2nd receptor potential. Summating potential. Some believe they can be used in the detection of certain end organ disorders and that they originate from OHCs and/or IHCs.

§                                               Latency: SP & nerve: 0-2ms.

§                                               Nerve and brainstem: 2-10ms

§                                               Thalamus & cortical: 50-300ms.

§                                               Late association cortex: >300 ms.

See the tables and illustrations of ABRs attached. Remember that the filter settings may be crucial for comparision with previously gathered/published data.

Record ABRs (averaged brainstem responses) from the vertex of the skull. Using a teflon-coated silver wire, place one end on the skull along the midline. The reference can be almost anywhere (on the animal). The filters should be set to: 3 - 3000 Hz.

The stimulus is the click (impulse). These potentials are quite small so that amplifications of 50,000 to 100, 000 are routinely used. You will need to insert a second amplifier in your signal line before it is sampled by the A/D. observe the signal on the oscilloscope to ensure that there is no clipping of the signal. In normal recordings, one has to be able to reject signal traces that are artifacts…. For example, muscle potentials. The goal is to utilize the full dynamic range of the A/D system that can cover ± 5V. So the signal amplitude at the output of the amplifier should cover the same range.

Begin with 70 dB SPL & average 1000 click responses.

If this doesn't yield anything try a higher SPL. Another variable is the number of averages. Try 5000. This of course will take longer. The Signal-to-noise ratio (S/N) improves by a factor of 2 for every increase of 4 in the number of averages.

Remember: there could be artifacts averaged into the final result if  proper” care is not exercised.

·       What might account for some of the differences?

·       Include an example of the ABR in your report.

·       Label the neural generators (probable sources) for the different waveforms.

·       Hand in: A set of curves with N1 responses indicated. Create a TH vs Frequency plot for CM and N1. Indicate the threshold of CM and N1 vs log (frequency).

At the end of the experiment: make an opening in the tympanic membrane about 2mm in diameter. Repeat the measurement of the N1 threshold curve. What changes occurred?

Write up this experiment as a short report. Do this individually. Use a word processor.  You can get a copy of this on the web at the class site.   This lets you copy sections, like the questions, eliminating retyping.

Ø     Provide an Introduction.

Ø     Methods. (describe the experimental setup. E.g. Filter settings, amplifiers, A/D, connections, software, etc.)

Ø     Results.

Ø     Discussion.

Ø     Addendum.

Ø     Repeat the questions in this exercise and give your answers in the report.